The long range goals of this project are to determine the responses of mammalian neurons to applied D.C. electrical fields and to understand the nature of the interaction of these fields with neurons. At present, there is no information about these matters although small D.C. currents have been reported to produce some functional recovery in mammals with transected spinal cords. Thus our first priority will be to determine if mammalian neurons respond to electrical fields in vitro and, if they do, then further experiments will help to provide a clearer understanding of the nature and mechanism of the responses. The results of these investigations should help assess the whole animal studies and may lead to insights for further experiments. Initial experiments will be done using PC 12 cells. These cells are derived from a rat pheochromocytoma, and in response to nerve growth factor (NGF) they stop dividing and differentiate into sympathetic-like neurons. As there may be some doubts about using a transformed cell line as a model for normal neuronal development or regeneration, some primary cultures of disaggregated neurons will be also used. A CNS preparation of neurons from mouse embryonic spinal cords will be one such culture. Peripheral neurons from mouse DRG and from mouse olfactory epithelium will be the other cultures studied. In all cases, small D.C. electrical fields will be applied in a way that eliminates possible secondary effects of the fields such as temperature changes, pH changes and nutrient or growth factor gradients. A computer-assisted system for analyzing the responses of the cells has been developed and using this it will be possible to decide, in a rigorous statistical way, if the growing neurites respond to electrical fields. In the cases where cells can be cultured on defined subtracts, the effect of different substrates on the responses to the electrical fields will be investigated. Questions about the mechanism of the interaction will be addressed by using antibodies to NGF receptor and the laminin receptor. The distributions of these receptors in the plasma membrane will be determined using quantitative fluorescence microscopy. The effect of an electrical field on these distributions will then be studied.
